Over the past few years the concern of safety of transgenic crop production has attracted significant attention among the scientific community and the public at large. Several reports have shown pollen of transgenic plants may be spread from the field plots. Most problematic are crop species having wild relatives capable of hybridizing with the transgenic crop. Species such as Brassicae or various woody plants are good examples of high-risk groups. Even if the potential problem at present has been restricted to plants, it is likely that similar problems will be encountered when animals, such as fish, become more serious targets of transgenic production. It is also likely that a wide range of modifications will be applied to transgenic plants in the future thus intensifying the need to prevent transgene escape.
While the potential risks to human or animal health of a particular transgene and its product can be tested and measured, the impact of gene escape is more complex to assess. On the other hand, the potential value of transgenic improvement of crops is so large that rather than banning the use of transgenes is it is more productive to find solution to prevent gene escape.
Due to the great concern of transgene escape, several approaches to handle the problems have been presented. The first approach comprises plants with engineered sterility. The second involves control of seed germination, also referred to as “terminator technology” launched by Monsanto (U.S. Pat. No. 5,723,765). Additionally there are reports of application of conditionally lethal factors, also called suicidal genes.
Several groups have studied the use of engineered male (or female) sterility. In the male sterility (MS) method pollination is prohibited, for example, by arresting mRNA synthesis. Pollination can be restored by expression of a RNAase inhibitor. The recovering factor (RF) in said case is carried by the pollinating line, i.e. the two elements, MS and RF, are present in two different plant individuals. Only a hybrid of these two plant individuals carries both the blocking and the recovering factors and is therefore fertile. Thus, the male sterility technique is aimed to aid hybrid seed production. However, male sterility does not prevent escape of the transgene into the environment, because the pollinated female (MS) plants are capable to produce hybrid seeds, which may shatter and stay behind in the field after harvesting.
Engineered fertility control has also been achieved in transgenic Brassica napus by expression of the RNAase gene under an anther tapetum specific tobacco promoter (De Block and Debrouwer, (1993) Planta, 189: 218-225). Expression of the RNAase gene in the tapetum cells of the anther kills the pollen at early stages of development. Pollination of the male-sterile. flowers with pollen of transgenic B. napus plants expressing the RNAase gene under the same promoter recovers the male sterility (MS) trait and the hybrid plants can produce normal fertile pollen with expression of the two genes. U.S. Pat. No. 5,750,867 discloses the maintenance of male-sterile plants. U.S. Pat. No. 5,767,374 discloses a similar method for female-sterile (FM) plants in which method the ribonuclcase gene is expressed in stamen cells of female parental plants and the killer gene expression in hybrid plants is restored by expression of the restorer gene coming from the pollinating parent line. The blocking gene is expressed in the female organs of the parent plant, while pollen remains fertile. The FM plants are intended to be pollinated by male sterile (MS) plants for production of hybrid seeds.
U.S. Pat. No. 5,728,926 discloses a method based on the expression of antisense mRNA of a gene vital for anther development. The antisense molecule is expressed at the same time with the sense molecule, both preferably driven by the same promoter. Concurrent expression of the sense and antisense orientations of the gene provides a silencing mechanism and thus prevents anther development. U.S. Pat. No. 6,013,859 discloses a method of hybridization. The blocking is provided by two consequent enzymatic reactions in the anthers or microspores. The pollinating parental line confers resistance to a selective marker (herbicide). U.S. Pat. No. 6,005,167 in turn describes a method for male or female sterility based on blocking, preferably by antisense technique, of the chalcone synthase expression in a developing anther or another part of the flower. Chalcone synthase is a key-enzyme in the flavonoid biosynthesis. Blocking the expression of this gene leads to unrecoverable blocking of fertilization.
U.S. Pat. No. 5,723,765 describes a method for arresting seed germination. The technique comprises activation of the inhibited blocking gene function through excision of a specific DNA sequence between the promoter and the blocking (terminator) gene by a specific Cre recombinase enzyme encoded by another gene placed under the Tet-repressed promoter. Seeds of transgenic plants not treated with tetracycline are capable of germinating under natural conditions. If the transgenic seeds are treated with tetracycline, the gene encoding for Cre recombinase is activated and it excises the DNA insertion between the LEA promoter and the toxin gene. Thus, the block is removed and the toxin activated. The toxin does not kill the plant immediately because the expression will be initiated only during late embryogenesis with the action of the LEA promoter.
The invention disclosed in U.S. Pat. No. 5,723,765 demonstrates inhibition of the development of seeds in the second generation. Without the mechanism of suppression, the ‘killer’ gene is activated during late stages of embryo development of the progeny and the seeds of the next generation do not germinate. The fundamental problem with said technique is that once the plants have been treated with tetracycline, i.e. the killer gene has been activated, they cannot be reused. Furthermore, if said transgenic plant carrying the tetracycline recoverable construct escapes into the environment, it is capable of germinating, growing to maturity, flowering and reproducing sexually. In other words, the transgene escape from transgenic plants is not prevented.
The technique described in U.S. Pat. No. 5,723,765 is often called “terminator” technique and it has encountered negative public attention because it gives the seed producing companies a possibility to control the market of transgenic seed production. Moreover, the technique does not prevent transgene escape.
Another related patent, U.S. Pat. No. 5,880,333 describes a method for regulating transgene expression through “receptor DNA cassettes” and chemical ligands activating the constructs. For example unrecoverable block (arrest) of embryo development has been achieved in Brassica napus by expression of the modified exotoxin A of Pseudomonas aeruginosa under napin promoter (Koning et al. 1992, Plant Mol. Biol., 18: 247-258). Pollen sterility has been achieved using diphtheria toxin A chain expression under lat52 promoter. Toxin expression leads to cell ablation in developing pollen (Twell, 1995, Protoplasma, 187: 144-154).
U.S. Pat. No. 5,498,533 describes the regulation of potato development by expression of sense and antisense constructs of the calmodulin gene. Expression of sense-oriented calmodulin gene increases shoot and tuber growth, whereas plants carrying antisense constructs exhibit decreased shoot and tuber growth. Therefore the expression of antisense calmodulin gene may be used as a factor blocking a physiological function.
In other words, various inventors have described different types of unrecoverable block of function systems intended to provide control of escape of transgenic plants. The main problem related to the above described methods and systems, is that they are unrecoverable. The seeds cannot be reused once the recovery system has been applied. A further limitation of some of the methods is that they require vegetative propagation, because fertilization or embryo development is arrested.
The object of the present invention is to provide a method and DNA-constructs for blocking a function. The blocked function is not recovered under natural conditions, but is capable of being recovered only when an external controllable treatment or intervention is applied. Thus, the present invention not only stops segregation and escape of trangene(s) into environment but also allows the farmer provided with the correct instructions to reuse his crop.
According to this disclosure the solution to the problem of transgene escape is achieved by providing a DNA construct preventing sexual reproduction in absence of an external treatment or intervention. Normal growth and sexual reproduction can be achieved by an external treatment or intervention per se or by applying a specific recovering DNA construct in the RBF system.